Bromoacetate reacts with histidine residues 12 and 119 at the active site of bovine pancreatic ribonuclease (RNase) much more rapidly than with free histidine. The mechanism of this facilitated alkylation was investigated by studying the dependence of the reaction on temperature and pH. RNase was treated with bromoacetate under pseudofirst-order conditions at 12, 25, 37, and 50 O C . The rate of inactivation of the enzyme showed a hyperbolic dependence on bromoacetate concentration, indicating formation of an enzyme-bromoacetate complex (K, = 41 m M at pH 5.5 and 25 "C). Two groups, one of which must be unprotonated and the other protonated, are required for carboxymethylation of RNase by bromoacetate. At 25 OC, the free enzyme exhibits macroscopic pK values of 4.7 and 6.3, and the enzymebromoacetate complex has pK values of 5.8 and 7.4. The ratio of products [N"-(carboxymethy1)histidine-1 19 RNase to N'-(carboxymethy1)histidine-12 RNase] formed in the reaction was 4.4 and was independent of temperature. Calculations based on this ratio and the microscopic pK values of histidines-1 19 and -12 determined by N M R titration suggest that the pH-independent alkylation of histidine-1 19 is about 8 times

The reaction of the imidazole group of histidine hydantoin with bromoacetate was studied as a model for carboxymethylation of histidine residues in proteins. pK values of 6.4 and 9.1 (25 degrees C) and apparent heats of ionization of 7.8 and 8.7 kcal/mol were determined for the imidazole and hydantoin rings, respectively. At pH values corresponding to the isoelectric points for histidine hydantoin, the rates of carboxymethylation at 12, 25, 37, and 50 degrees C were determined; the modified hydantoins were hydrolyzed to the corresponding histidine derivatives for quantitative amino acid analysis. At pH 7.72 and 25 degrees C, the imidazole tele-N was alkylated (k = 3.9 X 10(-5) M-1 s-1) twice as fast as the pros-N. The monocarboxymethyl derivatives were carboxymethylated at the same rate at the pros-N (k = 2.1 X 10(-5) M-1 s-1) but 3 times faster at the tele-N (k = 11 X 10(-5) M-1 s-1). The enthalpies of activation determined for carboxymethylation of the imidazole ring and its monocarboxymethyl derivatives were similar (15.9 +/- 0.7 kcal/mol). delta S for the four carboxymethylations was -25 +/- 2 eu. The electrostatic component of delta S (delta S es) was calculated from the influence of the dielectric constant on the reaction rate at 25 degrees C. delta S es was slightly negative (-4 +/- 1 eu) for mono- or dicarboxymethylations, indicating some charge separation in the transition state. The nonelectrostatic entropy of activation was -21 +/- 2 eu for all four carboxymethylations.